Present therapy for clinical management of brain injury, such as stroke, is directed toward maintaining blood flow to the brain and preventing further damage. In the case of an infarct or stroke, anticoagulants such as heparin and warfarin may be prescribed for short-term use to prevent blood clots from becoming larger. Aspirin or warfarin may also be used by certain stroke patients to prevent future strokes. Other therapy involves the application of thrombolytics for dissolving clots. With hemorrhagic stroke, drugs are administered to control brain swelling, high blood pressure and vasospasm.
It has been reported that extracellular levels of the excitatory amino acids (EAAs), glutamate and aspartate, increase 4-10 fold (Benveniste, H. et al., J. Neurochem. 1984 43:1369-1374) during or shortly following neuroinjury, resulting in indiscriminate and continuous activation of postsynaptic EAA receptors. This elevation of extracellular EAA levels is thought to be part of the periphenomena of most acute neuroinjury events, and may be one of the initial mechanisms in the cascade of events leading to cell damage. (Rothman and Olney, Trends Neurosci. 1987 10:299-302). This continuous EAA-mediated neurotransmission which results in cell death has been termed excitotoxicity (Olney, J. Neuropathol. Exp. Neurol. 1971 30:75-90). It has been reported that excessive activation of EAA receptors results in sustained elevated intracellular levels of Ca.sup.++ at the post-synaptic site, and that this increase in intracellular Ca.sup.++ activates numerous intracellular enzymes, including proteases, lipases and kinases (see FIG. 1) (Choi, Neurosci. Lett., 1985, 58:293-297). If unabated, combined and persistent activation of these intracellular mechanisms leads to loss of cellular integrity and eventual cell death.
EAA receptors have been grouped into four subtypes: (1) n-methyl-d-aspartate (NMDA); (2) kainic acid (KA); (3) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA); and (4) trans-amino-1,3-cyclopentanedicarboxylic acid (ACPD) (Marangos and Miller, 1991, in Adenosine and Adenine Nucleotides as Regulators of Cellular Function, ed. J. Phillis, p.360-365). Glycine has been reported to modulate NMDA receptor-mediated neurotransmission (Johnson, J. W. and Ascher, P., Nature 1987 325:529-531). It has also been reported that glycine is an obligatory component for NMDA receptor function (Kleckner and Dingledine, Science 1988 241:835-837).
Two different animal models of stroke are currently used in preclinical studies: global ischemia and focal stroke. Global ischemia results in complete loss of blood flow to the brain and complete loss of energy reserves and ion homeostasis. This model is generally more analogous to strokes occurring as a result of revival from a heart attack, cardiopulmonary arrest, strangulation or short-term interruption of blood flow to the brain.
In the global brain ischemia model the time period over which cell loss occurs has been reported to vary (Smith et al., Acta Neuropathologica, 1984 64:319-332; Kirino et al., Brain Res., 1982 239:57-69). In addition, it has been reported that there are regions of the brain which are selectively sensitive to ischemia-induced mechanisms. For example, it has been reported that cell death appears within hours in the hippocampal CA4 subregion, within a day in the striatum (Crain et al., Neuroscience, 1988 27:387-402), and over a period of 2-3 days within the hippocampal CA1 region.
Focal stroke is a better model than global ischemia for a majority of human strokes of thromboembolic origin or associated with the surgical procedures such as carotid endarterectomy or coronary artery bypass graft (CABG). A focal stroke is characterized by a gradient of blood flow from near total cessation in the central area of a blood vessel's territory of distribution to normal or increased flow at the periphery. Cell damage is confined to a region of the cortex within which a small core area is unsalvageable but a peripheral region (called the penumbra) is redeemable. Neuronal activity throughout the region is depressed, while ion homeostasis is maintained or intermittently disrupted in the penumbra region by periods of depressed neuronal activity, also known as waves of spreading depression.
It has been reported that adenosine inhibits EAA release (Burke and Nadler, J. Neurochem. 1988 51:1541-1551) (See FIG. 1). Adenosine acts at the level of the cell plasma membrane by binding to receptors. In both the brain and peripheral tissue these receptors have been designated as purinergic (P.sub.1) receptors (Burnstock, Pharmacol. Rev., 1972 24:509), which have been further classified into A.sub.1 and A.sub.2 receptor subtypes (Daly et al., Cell. Mol. Neurobiol. 1983 3:69-80). A.sub.1 and A.sub.2 receptor subtypes have been defined on the basis of their effect on cyclic adenosine 5'-monophosphate (cAMP) production and their structure activity relationships (Trevedi, et al., "Structure-Activity Relationships of Adenosine A.sub.1 and A.sub.2 Receptors," Adenosine and Adenosine Receptors, (Williams, M. 1990)). For example, A.sub.1 agonists are characterized by their ability to displace N-6 cyclohexyladenosine (CHA) specifically bound to the A.sub.1 receptor in membranes prepared from the cortex of rat brains. A.sub.2 agonists are characterized by their ability to displace N-ethylcarboxamido adenosine (NECA). Applicants have demonstrated that inhibition of EAA release in vivo results from A.sub.1 receptor activation. Adenosine agonists have affinity for both A.sub.1 and A.sub.2 receptors with some showing a greater affinity for A.sub.1 than A.sub.2 receptors. However, known A.sub.1 -specific agonists will react with A.sub.2 receptors when the A.sub.1 agonist concentration is sufficiently elevated so as to achieve a local concentration which is effective at the A.sub.2 receptor.
Several investigators have observed efficacy in the global ischemia and focal stroke models upon application of adenosine regulating compounds, adenosine agonists and transport inhibitors. The following investigations were carried out using a global ischemia model. Studies have examined compounds which increase adenosine levels by inhibition of adenosine deaminase (Phillis, J. W. and O'Regan M. H., Brain Res. Bull. 1989 22:537-540), purine nucleoside phosphorylase (Phillis, J. W. et al., Int. J. Purine & Pyrimidine Res. 1990 1:19-23) and xanthine oxidase (Phillis, J. W., Brain Res. Bull. 1989 23:467-470 and Helfman, C. and Phillis, J. W., Med. Sci. Res. 1989 17:969-970). Investigations with adenosine agonists have included cyclohexyl adenosine (CHA) (Von Lubitz DKJE et al., Stroke 1988 19:1133-1139: Daval, J. L. et al., Brain Res. 1989 491:212-226) 2-chloroadenosine (Evans, M. C. et al., Neurosci Letts. 1987 83:287-292) and L-phenylisopropyladenosine (Block G. A. and Pulsinelli, J. Cere. Blood Flow Metabol. 1987, 7(suppl 1):S258). The transport inhibitor propentofylline has also been reported to be efficacious in the global ischemia model (DeLeo J. et al., Neurosci Letts. 1988 84:307-311; DeLeo, J. et al., J. Cereb. Blood Flow Metab. 1987 7:745-751; DeLeo, J. et al., Stroke 19:1535- 1539: Hagberg, H. et al. in Pharmacology of Cerebral Ischemia, eds J. Krieglstein & H. Oberpichler, 1990, 427-437; Dux e. et al., Brain Res. 1990, 516:248-256). Another study examined adenosine agonist, R-phenylisopropyladenosine, in a focal stroke model and reported neuroprotection (Bielenberg G. W., J. Cere. Blood Flow Metabol. 1989, 9(supp 1):S645).
In one study, using a global stroke model, injections of 2-chloroadensine (2CLA) directly into the hippocampus were examined (Evans M. C., Swan, J. H. and Meldrum, B. S., Neurosci Letts. 1987 83:287-292). In these experiments efficacy was examined with iterative injections of 2CLA (a) immediately before a 10 minute period of ischemia and then at 4 and 10 hours into reperfusion, (b) at 1 minute, 4 and 10 hours into reperfusion, and (c) at 10 and 24 hours into reperfusion. It was reported that 2CLA protected against cell loss in protocols (a) and (b), but not in protocol (c).